1. Field of the Invention
The present invention relates to low dose multicomponent vaccines. More specifically, the invention relates to low dose multicomponent vaccines comprising a safe and immunogenically effective combination of: at least one protective antigen component from clostridial organisms, at least one protective antigen component from a non-clostridial organism and an adjuvant.
2. Brief Description of the Prior Art
Preparation and formulation of multicomponent vaccines have historically been complicated by physical and technological hurdles. Multicomponent vaccines of interest are those vaccines that contain as essential antigen components: one or more protective antigens from one or more organisms and an adjuvant. The protective antigen component can be in the form of a whole bacterial culture, a whole virus culture, a cell-free toxoid, a purified toxoid, or a subunit.
When one combines whole cultures of organisms (viruses or bacteria) in a formulation of multicomponent vaccines, the formulation would contain numerous antigens (hundreds to thousands). Some of these are protective antigens as mentioned above. Some of these antigens are detrimental to protection of the animals or cause reaction in the animals (xe2x80x9cdetrimental antigensxe2x80x9d). The detrimental antigens can interfere with the protective antigens by either physically or chemically blocking the active sites of the protective antigens. The interference prevents the protective antigens from protecting animals. Also, the detrimental antigens can produce negative responses such as local reactions, systemic reactions, anaphylaxis and/or immunosuppression in the animals. Therefore, the use of combinations of whole culture organisms can cause problems with efficacy or with animal reactivity. Animal reactivity produces localized reactions resulting in swellings or abscesses at the injection sites or a systemic response such as anaphylaxis that can result in death of the animal.
Aggravating the animal reactivity is the administration of multi-component vaccines to large animals (e.g., cattle) in high doses. The dose range has historically been from about 5 mL to 10 mL to allow incorporation of all of the protective antigens into one formulation. Illustratively, up to seven clostridial whole cultures or toxoids can be combined into a 5.0 mL dose of vaccine for administration to cattle. See, for instance, pages 319, 320, 321, 322, and 432 of the Compendium of Veterinary Products, Third Edition, 1995-1996). Also, 6 Clostridial whole cultures or toxoids have been combined with Hemophilus somnus in a 5.0 mL dose vaccines. See pages 191, 192, 319, 433, 490, and 1013 of the Compendium of Veterinary Products, Third Edition, 1995-1996). Reportedly, such vaccines demonstrate significant animal reactivity.
Animal reactivity that produces localized reactions (often called injection site lesions or blemishes) have become a matter of significant concern for the beef industry. Many scientific and lay articles since 1991 have addressed the concern with injection site lesions. See Stokka et al, J. Am. Vet. Med. Assoc., 1994, Feb. 1, 204(3): 415-9, Effertz, Beef Today, March 1991 and Beef Today, September 1992, Dittmer, CALF News Cattle Feeder, September 1992; Smith, FEEDSTUFFS, Aug. 24, 1992, and Hrehocik et al, dvm, September 1992. During the past several years, many scientific and lay articles have reported that injection site lesions are deleterious to the quality of beef. The injection site lesions must be cut out of the meat and discarded. This causes significant monetary loses to retailers, beef packers and feedlots. It has been estimated that 12-15% of prime beef cuts have some type of injection site lesion that must be trimmed away (Effertz, Beef Today, March 1991). This article attributes the main cause of the injection site lesions to 7-way clostridial vaccines. Additionally, there have been reports that up to 90% of cattle have injection site lesions in their carcass. Injection site lesions have been associated with: (1) the presence of many detrimental antigens or contaminants which are present in whole culture vaccines, (2) the adjuvants incorporated into such vaccines, (3) the method of administration of such vaccines (4) the large dose size of some of the multicomponent vaccines (5.0-10.0 mL), and (5) animal the reactivity of the protective antigen components of the vaccines.
Typically, clostridial vaccines are not highly purified because purification can be cost prohibitive. As one would realize, animal vaccine production must be necessarily economically effective if the vaccines are to enjoy widespread use. Therefore, highly purified animal vaccines are virtually cost prohibitive.
Somewhat related prior art involves two vaccines containing six clostridial whole cultures or toxoids administered in a 2.0 mL dose volume. See Compendium of Veterinary Products, Third Edition, 1995-1996, pages 133, 1183, 1184 and 1185 and the advertising brochure entitled xe2x80x9cALPHA-7(trademark)-JUST ONCExe2x80x9d. However, these vaccines do not include any additional component such as: additional clostridial component(s) or one or more non-clostridial component(s).
Antigenic components of clostridial vaccines were typically obtained by concentrating whole cultures of the bacteria. Concentration was accomplished by precipitating whole cultures with ammonium salts such as ammonium sulfate or concentrating such whole cultures via ultrafiltration. Both procedures are costly. Additionally, these procedures produce massive amounts of cells resulting in a high antigen mass that remains as an antigenic mass of solids in the product. Such a high antigenic mass would induce animal reactivity, particularly injection site lesions.
An even greater problem exists when one combines clostridial organisms with non-clostridial organisms such as Gram-negative bacteria, e.g., H. somnus and M. bovis and the Pasteurella spp. Many of these organisms are, in themselves, highly reactive and contain high levels of endotoxin that produce anaphylaxis. Also, their antigenic components supposedly cause interference. The high dose of the art-known combination of H. somnus and six clostridial components, i.e., a 5.0 mL dose volume can be the source of animal reactivity. In the case of non-clostridial viral formulations, the addition of clostridial components to these formulations can adversely affect viral epitopes. Consequently the viral components of the formulation may become non-efficacious.
Because of the severity of the Clostridial diseases and other disease complexes described herein, it is increasingly important that calves and young cattle entering feedlots as well as pregnant cows are properly vaccinated. The vaccines must contain protective antigens described herein. While one could administer each of the protective antigens in a monovalent vaccine, this mode of administration would require several vaccinations for each animal. This is impractical in a because: 1) handling animals for repeated vaccinations can result in undue stress and consequential diseases; 2) labor for performing such vaccinations is expensive compared to the profit obtained from each animal; 3) the more injection sites on an animal, the more potential for injection site reactions.
There is, therefore, a clear need for multicomponent vaccines containing many protective antigens that do not contain detrimental antigens and do not produce animal reactivity. By this invention, there are provided low dose multicomponent vaccines containing: protective antigen components of a clostridial organism(s) and at least one non-clostridial protective antigen component and an adjuvant, and the processes for making and using the vaccines.
This invention relates to a multicomponent vaccine comprising: a safe and immunogenically effective combination of protective antigen components from at least one clostridial organism, a protective antigen component from a non-clostridial organism and an adjuvant, wherein the vaccine is in a low dose volume. By xe2x80x9clow dosexe2x80x9d is meant dose volumes, including the adjuvant which are less than 5.0 mL and which do not adversely affect the protective antigen components or the animal post vaccination. Generally, an antigen is that which produces an antibody response against the antigen, which response is not necessarily protective. By the term xe2x80x9cprotective antigenxe2x80x9d is meant an antigen that produces an immune response and imparts protection to the animal. A vaccine containing such a protective antigen is characterized as xe2x80x9cimmunogenically effective.xe2x80x9d
Also, encompassed by the invention is a multicomponent vaccine for ruminants comprising: a safe and immunogenically effective combination of a protective antigen component from at least two and preferably six to seven clostridial organisms; a protective antigen component from a non-clostridial organism and an adjuvant, wherein the vaccine is in a low dose volume.
In the present embodiment of the invention, the multicomponent vaccine comprises a safe and immunogenically effective combination of an antigen component from one or more clostridial organisms; an antigen component from an organism selected from the group consisting of a Gram negative organism, a Gram positive organism, a virus, a parasite and a rickettsia and an adjuvant wherein the vaccine is in a dose size of 3.0 mL or less.
In a preferred embodiment of the invention, the multicomponent vaccine for ruminants comprises a safe and immunogenically effective combination of an antigenic component from six clostridial organisms, which are Clostridium chauvoei, Clostridium septicum, Clostridium novyi, Clostridium perfringens type C, Clostridium perfringens type D and Clostridium sordellii, an antigen component from H. somnus or M. bovis and an adjuvant, wherein the vaccine is in a dose size of 3.0 mL or less.
In another preferred embodiment of this invention, the multi-component vaccine for ruminants comprises: a safe and immunogenically effective combination of a protective antigen component from seven clostridial organisms which are Cl. chauvoei, Cl. septicum, Cl. novyi, Cl. perfringens type C, Cl. perfringens, type D, Cl sordellii, and Cl. haemolyticum; an antigen component from Haemophilus somnus or Moraxella bovis and an adjuvant, wherein the vaccine is in a dose size of 3.0 mL or less.
In another preferred embodiment of this invention, the multi-component vaccine for ruminants comprises: a safe and immunogenically effective combination of an antigen component from at least two clostridial organisms such as Cl. perfringens type C and Cl. perfringens type D; an antigen component from a virus such as an infectious bovine rhinotracheitis virus (IBRV) and an adjuvant, wherein the vaccine is in a dose size of 3.0 mL or less.
A particularly preferred embodiment of this invention includes a multicomponent vaccine for ruminants comprising: a safe and immunogenically effective combination of a protective antigen component from more than two clostridial organisms selected from the group consisting of Cl. chauvoei, Cl. septicum, Cl. novyi, Cl. perfringens type C, Cl. perfringens type D, Cl sordellii, and Cl. haemolyticum; protective antigen components from viruses which are selected from the group consisting of an infectious bovine rhinotracheitis virus (IBRV), a parainfluenza type 3 virus (PI3V), a bovine virus diarrhea virus (BVDV) and a bovine respiratory syncytial virus (BRSV) and an adjuvant, wherein the vaccine is in a dose size of 3.0 mL or less.
In another particularly preferred embodiment of the invention the multicomponent vaccine comprises: a safe and immunogenically effective combination of a protective antigen component from at least six clostridial organisms; a protective antigen component from a plurality of viruses and an adjuvant, wherein the vaccine is in a dose size of 3.0 mL or less.
The most preferred embodiment of the invention is a multi-component vaccine comprising: a safe and immunogenically effective combination of a protective antigen component from at least seven clostridial organisms; protective antigen components from at least four viruses and an adjuvant, wherein the vaccine is in a dose size of 3.0 mL or less.
Further encompassed by the invention is a method for producing a multicomponent vaccine comprising a safe and immunogenically effective combination of protective antigen components from clostridial organisms and a protective antigen component from a non-clostridial organism and an adjuvant wherein the vaccine is in a dose size of 3.0 mL or less, said method comprising: 1) identifying the protective antigen component of each organism by in vivo or in vitro methods; 2) quantitating the protective antigen components using antigen quantitation assays to provide the protective antigen component in an amount sufficient to produce a protective vaccine with the least antigenic mass; 3) identifying components of the organisms containing detrimental antigens by using the antigen quantitation assays and animal reactivity testing; 4) purifying the protective antigen components which contain detrimental antigens to remove the detrimental antigens; 5) selecting for each organism requiring inactivation, an effective inactivating agent which kills the organism without denaturing the protective antigen component; 6) selecting an effective adjuvant which produces enhancement of immune response without causing unacceptable animal reactivity for each component; 7) adjuvanting the protective antigen components sensitive to the effects of detrimental antigens organisms individually; 8) pooling all protective antigen components.
Also, encompassed by the invention is a process for administering the vaccines of the invention to ruminants.
By the present invention, it has been demonstrated that there is a significant difference in the size of injection site lesions in cattle vaccinated with: (1) a conventional 5.0 mL dose multicomponent clostridial product and (2) the low dose (2.0 mL) multicomponent vaccine of this invention. The area of the injection site lesion produced by the low dose vaccine is significantly smaller, post injection than the lesion produced by the conventional 5.0 mL dose vaccine. The low dose multicomponent vaccine produced injection site lesions in an insignificant number of cattle as compared with the conventional vaccine.
In accordance with the invention it has been discovered that in the preparation of multicomponent vaccines such as those containing seven clostridial organisms, one can: identify and reduce the required antigenic mass and combine it with a compatible adjuvant to produce a low dose, safe and immunogenically effective vaccine. This discovery is the basis of the inventive concept described herein. According to this inventive concept, the skilled artisan can combine: protective antigen components from the clostridial organisms and non-clostridial organisms, and an adjuvant in a low dose volume, and safely administer it to ruminants to protect them against diseases described more fully hereunder.
More specifically, the invention relates to a multicomponent vaccine comprising a safe and immunogenically effective combination of: an antigen component from one or more clostridial organisms; an antigen component from a non-clostridial organism selected from the group consisting of a Gram negative organism, a Gram positive organism, a virus, a parasite and a rickettsia and an adjuvant, wherein the vaccine is in a dose size of 3.0 mL or less. Non-limiting examples of the clostridial organisms and diseases in ruminants are as follows:
Clostridium chauvoei causes the disease blackleg. This organism, like all Clostridial organisms, produces spores that can survive in soil for years and, during this time, can infect susceptible animals (cattle and sheep) which ingest them. Blackleg is an acute, infectious but noncontagious, disease of cattle and sheep characterized by gaseous tissue swelling, usually in the heavy muscles. The organism enters cattle or sheep via feed or cuts or by shearing, docking, or castration. The onset of the disease is quite sudden. Body temperature rises rapidly and muscular stiffness, depression and reluctance to move are prominent. When infection is extensive, death often occurs within 16-72 hours. Treatment of sick animals is futile since there is often permanent damage done to their meat.
Clostridium septicum causes the disease of malignant edema, or gas gangrene, a rapidly extending edematous swelling, in subcutaneous tissues of cattle. The disease is characterized by gangrene and gaseous swelling surrounding a wound. Incidence of the disease often follows castration, dehorning, accidental puncture wounds and lacerations, abortions, and vaccination with unclean needles. The incubation period is short and death occurs within 12 to 48 hours. Death is primarily caused by toxins released by multiplying organisms after infection occurs. As with Cl. chauvoei, it is impractical to treat the animals.
Clostridium novyi causes the condition of black disease or infectious necrotic hepatitis which is an acute infectious disease of cattle and sheep. The causative spore-forming organism may enter cattle through the digestive tract, lungs or wounds. In areas where liver flukes are endemic, Cl. novyi is especially dangerous because the organism will multiply in damaged areas resulting from the migration of liver flukes. The organism multiplies rapidly and produces a highly fatal exotoxin causing toxemia and death. Death is usually sudden with no well-defined signs. Because of the rapidity of death, treatment is not practical.
Clostridium sordellii causes a disease similar to Cl. novyi and Cl. septicum. The organism is an inhabitant of the soil and of the animal intestine. Most infections by the organisms are associated with wounds or liver flukes. Lesions at the site of the infection progress rapidly, followed by fever, depression and edema that is similar to that produced in Cl. novyi infections. A rank odor is detected in diseased tissues. Death is also sudden indicating that treatment is not practical.
Clostridium perfringens types B, C, and D are found as spores in the soil but are also parts of the normal intestinal flora of healthy animals. Under favorable conditions, such as when animals are being fed high protein diets in feedlots, the organisms multiply rapidly in the intestines. They produce lethal toxins which kill infected animals. Cl. perfringens type B causes sudden death in cattle and lambs. Cl. perfringens type C produces an acute hemorrhagic enteritis in calves, lambs, piglets and older cattle and sheep on high-energy feeds. Cl. perfringens type D causes overeating disease in feedlot cattle unaccustomed to high-energy concentration rations. All of the syndromes produced by the various types of Cl. perfringens have rapid onset and result in death before the animals can be effectively treated.
Clostridium tetani causes tetanus that can afflict all mammals.
The disease results from organisms entering their body via puncture wounds. As the organisms multiply, toxins which affect the central nervous system are produced. Infected animals become stiff, have difficulty swallowing and breathing, and are afflicted with spasmodic contractions of the musculature. While treatment with antitoxin is viable, it is extremely expensive and cost inefficient.
As set forth above, the non-clostridial organism can be selected from the group consisting of: a Gram negative organism, a Gram positive organism, a virus, a parasite and a rickettsia. The following is a non-limiting illustration of the Gram negative organisms.
Haemophilus somnus (H. somnus) is an organism that causes a complex of disease conditions found mainly in feedlot cattle The disease is, also, found in dairy and pasture cattle. This organism can cause a thromboembolic meningoencephalitis (TEME), a respiratory tract disease, reproductive diseases and a general septicemia. It is a non-motile, rod-shaped bacterium which is difficult to isolate and is most likely spread by respiratory secretions and discharges. Its incubation period is two to seven days. Infected animals can be treated successfully with antibiotics if they are treated early enough in the course of the disease. Unfortunately, once the infection becomes systemic, antibiotic effectiveness is decreased. Vaccination is the best method for protecting a herd of cattle from these H. somnus-induced diseases. The fact that H. somnus is a Gram-negative organism, and therefore contains endotoxin, renders the formulation of a non-reactive vaccine difficult. Moraxella bovis (M. bovis) is a Gram-negative organism that causes pink-eye in cattle. This disease is often chronic in herds of cattle and causes cattle to develop keratoconjunctivitis, with blindness a sequelae, after a period of time. Treatment is expensive as it must be continued for long periods of time. M. bovis has the potential to cause anaphylaxis and/or severe local reactions.
Campylobacter fetus is a Gram-negative organism that causes a venereal disease transmitted during breeding. Although the disease is often subclinical, it causes temporary infertility, irregular estrous cycles, delayed conception and, occasionally, abortion in cows.
Leptospira spp. infect and localize in the kidneys and are shed in the urine. Infection with Leptospira spp. can cause anemia, bloody urine, fever, loss of appetite and prostration in calves. Infection is usually subclinical in adult cattle. Infected pregnant cows, however, often abort, and dairy cows may exhibit a marked decrease in milk production. There are at least six major serovars in the species L. interrogans (L. pomona, L. canicola, L. grippotyphosa, L. icterohaemorrhagiae, L. hardjo, and L. bratislava),
Pasteurella haemolytica and Pasteurella multocida are causative agents of bovine pneumonia in feedlot cattle and young calves. They are the most significant components of the shipping fever complex and induce clinical pneumonia in cattle which are predisposed to infections with: infectious bovine rhinotracheitis, parainfluenza type 3 virus, bovine respiratory syncytial virus or bovine virus diarrhea virus. Infectious bovine rhinotracheitis virus causes a severe respiratory infection of cattle, specifically in feedlot conditions. The disease is characterized by: high temperature, excessive nasal discharge, conjunctivitis and ocular discharge, inflamed nasal mucosa, increased rate of respiration, coughing, loss of appetite, depression and/or reproductive failure in cattle. Infection with this virus often predisposes cattle to bacterial infections that cause death.
Parainfluenza type 3 virus (PI3) usually causes a localized upper respiratory tract infection, producing elevated temperatures and moderate nasal and ocular discharge. Although clinical signs of PI3 are typically mild, this infection weakens the respiratory defenses and allows replication of other pathogens, particularly Pasteurella spp.
Bovine virus diarrhea (BVD) is a major cause of abortion, fetal resorption or congenital fetal malformation. If susceptible cows are infected with non cytopathic BVD virus during the first trimester of pregnancy, their calves may be born persistently infected with the virus. Exposure of those calves to certain virulent cytopathic BVD virus strains may precipitate BVD-mucosal disease. Clinical signs of this disease include loss of appetite, ulcerations in the mouth, profuse salivation, elevated temperature, diarrhea, dehydration and lameness. The disease usually affects feedlot cattle.
Bovine respiratory syncytial virus (BRSV) infects cattle of all ages and causes: rapid breathing, coughing, loss of appetite, discharge from the nose and eyes, fever and swelling in the cervical area. In an acute outbreak, death may follow 48 hours after the onset of signs.
The following is a non-limiting illustration of the parasites that are employed herein.
Neospora spp. have been recently isolated form aborted fetuses. These organisms are parasites which have been proposed as a major cause of abortion in pregnant cows throughout the world. If this proves to be correct, a vaccine for protection of pregnant cattle against Neospora spp. could be a requirement in the future.
In accordance with the invention, clostridial organisms can be
selected from the group consisting of: Cl. chauvoei, Cl. septicum, Cl. novyi, Cl. perfringens type C, Cl. perfringens type D, Cl sordellii, and Cl. haemolyticum. Preferably, the protective antigen of the clostridial component is derived from six to seven clostridial organisms.
The non-clostridial protective antigen component can be selected from the group consisting of Gram negative bacteria, Gram positive bacteria, viruses, parasites, rickettsia and a combination thereof. Non-limiting examples of the Gram negative organisms can be selected from the group consisting of: H. somnus, M. bovis, E. coli, Salmonella typhimurium, Pasteurella hemolytica, Pasteurella multocida, Campylobacter fetus, Leptospira spp and a combination thereof. Preferred herein are H. somnus and M. bovis. 
Non-limiting examples of the Gram positive organisms are Clostridium tetani, Bacillus anthracis, Listeria monocytogenes, Actinomyces pyogenes and a combination thereof.
Non-limiting examples of the virus can be selected from the group consisting of: infectious bovine rhinotracheitis (IBRV), parainfluenza virus type 3 (PI3V), bovine virus diarrhea virus (BVDV) bovine respiratory syncytial virus (BRSV) and a combination thereof.
Non-limiting examples of the parasites are Neospora spp., Tritrichimonas foetus, Cryptosporidia spp. and a combination thereof.
A non-limiting example of the rickettsia is Ehrlichia bovis. 
In accordance with the invention, the clostridial and non-clostridial protective antigen components can be in the form of: inactivated or modified live whole cultures, toxoids, cell-free toxoids, purified toxoids, subunits or combinations thereof.
Adjuvants useful herein are by definition chemical compounds added to vaccines to enhance the production of an immune response by the animal receiving the vaccine. Most adjuvants function by: (1)producing an irritation at the site of injection causing leukocytes (immune cells) to infiltrate the area, and/or (2) by producing a depot effectxe2x80x94holding the antigen(s) at the injection site for as long as possible. If infiltration of leukocytes to the injection site is extensive, swelling and injection-site lesions will occur. Such leukocytes carry the antigens from the vaccine to cells within the immune system (of the vaccinated animal) which can produce a protective response. Some newer polymer adjuvants function by encapsulating antigens and releasing them slowly over a period of weeks or months. These newer adjuvants can help in protecting antigens from interference and are generally less likely to cause extensive infiltration of leukocytes to the injection site. In accordance with the invention, the adjuvants can be selected from the group consisting of: oil-in-water, water-in-oil, Al(OH)3, Al2(SO4)3, AlPO4, extracts of bacterial cell walls (Mycobacterium, Propionibacterium, etc.), extracts of plants (acemannan, saponin or QUIL A), polymers, including block copolymers, liposomes and combinations thereof. Preferred herein are adjuvants that function by encapsulating antigens and releasing them slowly over a period of weeks or months Preferably, the adjuvants are polymers, including block copolymers (alternately referred to herein as polymer adjuvants. A specific example of the preferred adjuvant is carbopol. Generally, the more effective the adjuvant is, the more irritating it is and the more likely it is to cause an animal reaction. It is a distinct feature of the invention that effective adjuvants can be formulated with the protective antigens to produce vaccines that are safe and effective.
It is also a distinct feature of the invention that a multicomponent vaccine for ruminants would include all the required protective antigen components and adjuvant, in a low dose. In essence, fewer than five protective antigens from each organism would be required to make a vaccine immunogenically effective. However, a vaccine containing only the protective antigens would be essentially a very pure vaccine. Because of the high purity of the antigens, it would be difficult adjuvant them with commonly used adjuvants. The pure antigen would require adjuvants that are different from the typical adjuvants. Therefore, a commercial scale production of clostridial vaccines containing very pure protective antigen components would be technically difficult. At any rate, the preparation of a very pure animal vaccine on a commercial scale is prohibitive because of the cost of purification.
In accordance with the invention, individual components of the multicomponent vaccines described herein can be formulated with protective antigens derived from: whole culture bacteria, whole culture viruses, cell-free toxoids, purified toxoids and/or subunits. Whole cultures contain numerous antigens. Some of the antigens impart protection (protective antigens), some produce negative response (detrimental antigens) and some are essentially neutral (neutral antigens). Subunits can be obtained from the organisms themselves by conventional methods such as: centrifugation, ultrafiltration, and extraction with detergents or organic solvents. Alternately, the subunits can be produced by recombinant technology and expressed in live vectors or other organisms and isolated and purified. It would be understood that protective antigen components may contain few to many antigens at least one of which is protective or immunogenically effective.
In the preparation of the vaccine of the invention, one can incorporate protective antigen components from a plurality of organisms. This occasions the likelihood of one protective antigen component interfering with another. This is particularly the case if the protective antigens are derived from clostridial organisms. The interference may result from: (1) physical masking or hiding of an active site of one protective antigen component by another, (2) aggregation or agglomeration of one or more protective antigen components so that one or more active sites are hidden from the immune system, (3) chemical interaction wherein there is a change in the active site of one protective antigen component by another. The latter change can result from a toxic effect, chemical binding or a conformational change in a critical portion of an active site.
It is a distinct feature of the invention that the effects of the detrimental antigens can be avoided by the process of the invention. The process comprises: using specialized procedures for identifying the protective antigen components; quantitating the protective antigen components; identifying those protective antigen components that contain detrimental antigens; purifying those protective antigen components that contain detrimental antigens to remove such detrimental antigens; selecting adjuvants that produce the necessary enhancement of the immune response without causing unacceptable reactivity and protect against interference; individually adjuvanting the protective antigen components that are sensitive to the effects of detrimental antigens; pooling the various protective antigen components into a low dose volume vaccine.
In the preparation of the multicomponent vaccines, the inventors employ adjuvant that protect the active sites of the various protective antigen components. In effect, the adjuvants interact with targeted protective antigens, and not other antigens. As would be realized, the selection of an adjuvant is critical. The adjuvant must be one that is potent enough to produce significant enhancement of the immune response without producing unacceptable local or systemic reactions. The term xe2x80x9cproduce significant enhancement of the immune responsexe2x80x9d refers to stimulation of the immune system such that protection of the host animal results from vaccination. Additionally, the adjuvant must reduce or prevent the interference with the protective antigens. An adjuvant that encapsulates antigens is preferred. This characteristic is usually associated with polymer- or block copolymer-type adjuvants. The preferred adjuvant for this invention is one containing xe2x80x9cCARBOPOLxe2x80x9d or the equivalent thereof.
An integral part of the invention is the use of a specified test method for antigen quantitation of the protective antigen components. Illustratively, the test method for quantitation of a clostridial protective antigen component involves injection of mice with combinations of antigen and specific antisera. The test method is referred to herein as xe2x80x9ca combining power testxe2x80x9d. The resultant measurement of antigen is designated as xe2x80x9ccombining power unitxe2x80x9d (CPU). The CPU test, developed in accordance with the invention, is an integral part of the formulation of combination clostridial products. The test comprises adding varying volumes of test material to a series of tubes. The total volume of test material in each tube is brought to 1.0 mL using Peptone Sodium Chloride Diluent [8.5 gm Sodium Chloride and 10 gm Bactone Peptone/liter (PND)]. One half milliliter of PND containing one International Unit of antitoxin, obtained from the clostridial organism being tested, plus enough excess antitoxin to neutralize approximately 100 MLD of toxin, is added to each tube. The tubes are mixed and 18 to 20 gm mice are inoculated intravenously with 0.5 mL from each tube. The mice are observed for 48 hours and death is recorded. The CPU of the test material is calculated as follows:       CPU    ⁢          /        ⁢    mL    =            Reciprocal      ⁢              xe2x80x83            ⁢      of      ⁢              xe2x80x83            ⁢      the      ⁢              xe2x80x83            ⁢      dilution      ⁢              xe2x80x83            ⁢      of      ⁢              xe2x80x83            ⁢      the      ⁢              xe2x80x83            ⁢      toxoid      xc3x97      2                                            Smallest            ⁢                          xe2x80x83                        ⁢            volume            ⁢                          xe2x80x83                        ⁢            of            ⁢                          xe2x80x83                        ⁢            the            ⁢                          xe2x80x83                        ⁢            above            ⁢                          xe2x80x83                        ⁢            dilution                                                            which            ⁢                          xe2x80x83                        ⁢            kills            ⁢                          xe2x80x83                        ⁢            100            ⁢            %            ⁢                          xe2x80x83                        ⁢            of            ⁢                          xe2x80x83                        ⁢            inoculated            ⁢                          xe2x80x83                        ⁢            mice                              
Other test methods that produce substantially the same results as described herein are encompassed by the claimed invention. Non-limiting examples of other test methods can be ELISA assays and liquid chromatography, which quantitate antigens directly in vaccines. In accordance with the foregoing, the skilled artisan can employ the required CPU/mL or the equivalent Elisa antigen quantitation unit to ascertain the value of the amounts of the antigenic components that are useful in making and using the vaccines of the invention.
The inventors have unexpectedly found that multicomponent vaccines containing a plurality of clostridial protective antigen components plus at least one non-clostridial protective antigen component and an adjuvant in a low dose volume can be produced by: identifying the protective antigen component of each organism by in vivo or in vitro methods; quantifying the protective antigen components during formulation and manufacture of the vaccine, using antigen quantitation assays described above to provide the protective antigen component in an amount sufficient to produce a protective vaccine with the least antigenic mass; identifying the antigenic components of the organisms which contain detrimental antigens by using the antigen quantitation assays and animal reactivity testing; purifying the protective antigen components which contain detrimental antigens to remove such antigens; selecting the inactivating agent for each organism requiring inactivation such that the organism is killed without denaturing the protective antigen component; selecting an adjuvant for each protective antigen component that requires an adjuvant by evaluating the adjuvant""s ability to enhance the immune response to the specific protective antigen component without causing unacceptable animal reactivity; adjuvanting, individually, the protective antigen components that require such adjuvanting; pooling the protective antigenic components into a low dose vaccine that imparts protection to animals to which the vaccine is administered. By this method, one can produces a commercially-viable, cost effective safe, immunogenically effective multicomponent vaccine. The multicomponent vaccine contains a combination of: one or more clostridial protective antigen components with one or more non-clostridial protective antigen components and an adjuvant within a low dose volume of 3.0 mL or less. The use of multicomponent vaccines, i.e., commercial scale vaccines of this infection, do not produce significant injection-site lesions upon subcutaneous or intramuscular administration.
The following is a specific description of the invention that is intended to assist those skilled in the practice of the invention. More specifically, the description relates to the characterization of the antigenic components and the manner in which they are formulated, including inactivation and adjuvanting.
Cl. chauvoei protective antigens have been found by the inventors to be associated with cells. These protective antigens are not found in proteinaceous material excreted into the culture supernatant while the organism is being grown in fermenters. It has also been found that the Cl. chauvoei protective antigen component does not interfere with other protective antigen components in the multicomponent clostridial vaccine. Therefore, a whole cell bacterin or a cell extract can be used. The whole cell bacterin or cell extract may be inactivated with formaldehyde (0.05-1.5%), Betapropriolactone (BPL) at 0.05 to 0.3% or Binary ethyleneimine (BEI) at 0.05 to 0.3%. After inactivation, this component must be adjuvanted separately. If BPL or BEI are used for inactivation they must be neutralized prior to adjuvanting. Adjuvants which enhance this C protective antigen component are Al(OH)3, oils, saponin, QUIL A, block co-polymers and polymers such as xe2x80x9cCARBOPOLxe2x80x9d. Oil adjuvants can be used as co-adjuvants with polymers. CARBOPOL is more preferred and is added to the inactivated whole culture at a low pH. The pH is then adjusted upward to approximately 7.0 with, say, sodium hydroxide (NaOH). This pH adjustment step allows for the protective antigen components of the Cl. chauvoei to become encapsulated in the polymer adjuvant. Without being bound to any particular theory of the invention, it is believed the Cl. chauvoei antigens are released over a period of several weeks. Because of the slow release, these antigens do not cause the typical animal reaction. The long-term release causes an enhanced immune response by the vaccinated animal.
The protective antigen component of Cl. septicum is associated both with the cell and with a toxin. The toxin is secreted into a supernatant while the organism is growing. Therefore, this protective antigen component is derived from the cell and supernatant. Apparently, Cl. septicum does not interfere with other protective antigen components in multicomponent clostridial vaccines containing non-clostridial protective antigen components. The whole cell bacterin or cell extract can be inactivated with formaldehyde (0.05-1.5%), BPL (0.05-0.3%) or BEI (0.05-0.3%). After inactivation, this protective antigen component must be adjuvanted separately. When BPL or BEI are used for inactivation, they must be neutralized before adjuvanting. Adjuvants that enhance this protective antigen component can be: Al(OH)3, oils, saponin, QUIL A, block co-polymers and polymers such as CARBOPOL. Oil adjuvants can be used if combined as co-adjuvants with polymers. The preferred adjuvant are the polymer adjuvant. Preferably, the adjuvant is added to the inactivated whole culture at a low pH. Then the pH is adjusted upward to approximately 7.0 with NaOH. This pH adjustment step increases the pH from approximately 5.0 to 7.0 during which the antigens of the Cl. septicum become encapsulated in the CARBOPOL. The resulting vaccine does not cause the typical animal reactivity but releases the Cl. septicum antigens over a period of several weeks. This mode of release causes an enhanced immune response by the vaccinated animal.
The protective antigen component of Cl. novyi, is believed by the inventors to be associated with a cell protein, and a toxin that is excreted into a supernatant. Therefore, this protective antigen component is derived from both the cell and supernatant, in either concentrated or non-concentrated form. Apparently, the protective antigen of the Cl. novyi does not interfere with other protective antigen components in multicomponent clostridial vaccines when combined with non-clostridial protective antigen components. The whole cell bacterin or cell extract may be inactivated with formaldehyde (0.05-1.5%), BPL (0.05-0.3%) or BEI (0.05-0.3%) and must be adjuvanted separately. If BPL or BEI is used, it must be neutralized before adjuvanting. Adjuvants that enhance this protective antigen component are Al(OH)3, oils, saponin, QUIL A, block co-polymers and polymers such as CARBOPOL. Oil adjuvants can be used if combined as co-adjuvants with polymers. The CARBOPOL polymer adjuvants are preferred. The polymer adjuvant is added to the inactivated whole culture at a low pH. Then the pH is adjusted upward to approximately 7.0 with NaOH. This pH adjustment step increases the pH from approximately 5.0 to 7.0 during which the antigens of the Cl. novyi become encapsulated in polymer. The resulting vaccine does not cause the typical animal reactivity but releases the Cl. novyi antigens over a period of several weeks. The long-term release causes an enhanced immune response by the vaccinated animal.
The protective antigen component of Cl. sordellii is believed to be associated with a toxin that is secreted into the supernatant as the culture is growing. Therefore, this protective antigen component is derived from the supernatant. This protective antigen component is typically concentrated via ultrafiltration through a 10,000 dalton molecular weight (MW) cartridge before adjuvanting. The Cl. sordellii toxin may be inactivated with formaldehyde (0.05-1.5%), BPL (0.05-0.3%) or BEI (0.05-0.3%) prior to adjuvanting, and must be adjuvanted separately. If BPL or BEI is used for inactivation, it must be neutralized before adjuvanting. Adjuvants that enhance this protective antigen component are Al(OH)3, oils, saponin, QUIL A, block co-polymers and polymers such as CARBOPOL. Oil adjuvants can be used if combined as co-adjuvants with polymers. The polymer adjuvant is are preferred. The CARBOPOL polymer adjuvant is added to the inactivated whole culture at a low pH. Then the pH is adjusted upward to approximately 7.0 with NaOH. This pH adjustment step increases the pH from approximately 5.0 to 7.0 during which the antigens encapsulated in polymer adjuvant. The resulting vaccine does not cause the typical animal reactivity but releases the Cl. sordellii antigens over a period of several weeks. The long-term release causes an enhanced immune response by the vaccinated animal.
The protective antigen components of Cl. perfringens types C and D are known to be toxoids that are excreted by the cells. Because they cross-protect against Cl perfringens type B, these protective antigen components only need to contain cell-free supernatant containing inactivated toxin (toxoid). These two components are considered to represent 3 components (B,C, and D). In formulations of a multicomponent clostridial vaccine, one may use Cl. perfringens types C and D protective antigen components that contain cells or have the cells removed therefrom (cell free toxoid). Before the removal of the cells, the whole culture is harvested from the fermenter and inactivated with formaldehyde (0.5-1.5%), BPL (0.05-0.5%) or BEI (0.05-0.5%) and before adjuvanting. The cells can be removed by, say, filtration or centrifugation, In either case, the respective antigens must be adjuvanted separately. If BPL or BEI is used for inactivation, it must be neutralized before cell removal. Adjuvants which enhance this protective antigen component are Al(OH)3, oils, saponin, QUIL A, block co-polymers and polymers such as CARBOPOL. Oil adjuvants can be used if combined as co-adjuvants with polymers. Preferred here is the polymer adjuvant. The CARBOPOL adjuvant is added to the inactivated whole culture at a low pH. Then the pH is adjusted upward to approximately 7.0 with NaOH. This pH adjustment step increases the pH from approximately 5.0 to 7.0. During this increase the protective antigen components of the Cl. perfringens become encapsulated in the polymer adjuvant.
The protective antigen component of Cl. haemolyticum is believed to be both cell-associated and excreted as a toxin into the supernatant. Therefore, this protective antigen component contains antigens from the cells and supematant. Because of its high cell mass, this protective antigen component can cause interference with other protective antigen components of a multicomponent clostridial vaccine. Typically, this protective antigen is concentrated by, say, ultrafiltration with a 10,000 molecular weight cartridge before adjuvanting. The Cl. haemolyticum whole culture can be inactivated with formaldehyde (0.05-1.5%), BPL (0.05-0.3%) or BEI (0.05-0.3%) before concentration. The inactivated, concentrated material must be adjuvanted separately. If BPL or BEI are used for inactivation, it must be neutralized prior to adjuvanting. Adjuvants which enhance this protective antigen component are Al(OH)3, oils, saponin, QUIL A, block co-polymers and polymers such as CARBOPOL. Oil adjuvants can be used if combined as co-adjuvants with polymers. Preferred herein is the polymer adjuvant. The CARBOPOL adjuvant is added to the inactivated whole culture at a low pH. Then the pH is adjusted upward to approximately 7.0 with NaOH. This pH adjustment step increases the pH from approximately 5.0 to 7.0. During the increase, the protective antigen components of the Cl. haemolyticum become encapsulated in polymer adjuvant. The resulting vaccine does not cause the typical animal reactivity but releases the Cl. haemolyticum antigens over a period of several weeks. The long-term release causes an enhanced immune response by the vaccinated animal.
With the foregoing description and the examples to follow, it would be within the purview of the skilled artisan to make and use the low dose, multicomponent vaccines of the invention. In the practice of the invention, the multicomponent, low-dose vaccines can be administered subcutaneously or intramuscularly to protect animals without causing significant injection-site lesions.
This and other aspects of the invention are further illustrated by the following non-limiting examples.